So-called modulation contrast microscopy (Hoffman modulation contrast) is a known microscopy method disclosed in Patent document 1. Manufacturers build contrast microscopes on the basis of the principles disclosed in Patent document 1.
Patent document 2 discloses a modulation contrast microscope that is based on the principles of Patent document 1. The modulation contrast microscope comprises a light source, a condenser lens, an aperture disposed in the front focal position of the condenser lens, an objective lens, and a modulator disposed at a position substantially conjugate with the aperture and having a region of transmittance T(%), wherein the microscope is configured so as to satisfy the conditions of formula (1) and formula (2):1.05<|(Mo′×fc)/(Mc′×fr)|<1.4  (1)8<T<25  (2)
In formula (1) and formula (2), Mo′ denotes the size, in the radial direction, of a region of transmittance T within a plane perpendicular to the optical axis in which the modulator is arranged, Mc′ denotes the size of the aperture in the radial direction, centered around the microscope optical axis, fc is the focal distance of the condenser lens, and fr is the focal distance of the optical system from the surface of a specimen up to the modulator.
In addition to formula (1) and formula (2), Patent document 2 discloses also an instance where the microscope further satisfies the conditions of formula (3):0.2R<D<0.6R  (3)
In formula (3), R denotes the size of the effective radius of the plane at which the modulator is disposed, and D denotes the distance from the region of transmittance T from the microscope optical axis.
Patent document 1: Japanese Patent Application Laid-open No. 51-128548
Patent document 2: Japanese Patent Application Laid-open No. 2003-131139
As indicated in Patent document 2, so-called ICSI (intracytoplasmic sperm injection) has been used in recent years as an in-vitro fertilization technique for the treatment of infertility.
To inject a sperm into an egg cell, the normal procedure of ICSI often involves suctioning first a sperm from the tail of the latter. Sperm of good morphology must be selected, and it must be ensured that sperm are pipetted from the outmost end of the tail so as not to damage the sperm, in order to increase the fertilization success rate.
Under the modulation contrast microscopy conditions set forth in Patent document 1 and Patent document 2, however, contrast is weak for sperm, which in ICSI is as important as the egg. Contrast is weak, in particular, at the tail end of the sperm. The above methods are problematic in that, as a result, it is difficult to distinguish the tail end portion within the small form of sperm, and it is thus difficult to select sperm of good morphology, and to pipette sperm from the outmost end of the tail.
In particular, Patent document 2 sets forth “viewing is thus optimized, with good balance between resolution and contrast, but without excessive contrast, also in fine portions such as the zona pellucida and granules of the egg, by setting the partial aperture and the length of the region of transmittance T to lie within the range of formula (1) above”. Patent document 2, however, does not set out any rationale for the transmittance T in formula (2), nor does it make any mention of contrast in sperm, in particular the tail.